Efficient production of bioactive proteins and peptides is a primary function of the biomedical and industrial biotechnology industry. Bioactive peptides and proteins are used as curative agents in a variety of diseases such as diabetes (insulin), viral infections and leukemia (interferon), diseases of the immune system (interleukins), and red blood cell deficiencies (erythropoietin), to name a few. Additionally, large quantities of proteins and peptides are needed for various industrial applications including, but not limited to pulp and paper industries, textiles, food industries, personal care and cosmetics industries, sugar refining, wastewater treatment, production of alcoholic beverages and as catalysts for the generation of new pharmaceuticals.
In biomedical-related fields small peptides are sometimes used as linkers for the attachment of diagnostic and pharmaceutical agents to surfaces (see U.S. Pat. App. Pub. No. 2003/0185870 to Grinstaff et al., and U.S. Pat. No. 6,620,419 to Linter). In the field of personal care, small peptides have been used to couple benefit agents to body surfaces such as hair, skin, nail, and teeth (U.S. Pat. Nos. 7,220,405; 7,309,482; 7,129,326; and 7,285,264; U.S. Pat. App. Pub. Nos. 2002/0098524; 2005/0112692; 2005/0226839; 2007/0196305; 2006/0199206; 2007/0065387; 2008/0107614; 2007/0110686; and 2006/0073111; and Int'l App. Pub. Nos. WO2008/054746; WO2004/048399, and WO2008/073368).
Peptides may be prepared by chemical synthesis or isolated from natural sources. However, these methods are often expensive, time consuming, and characterized by limited production capacity. The preferred method of producing large quantities of peptides or proteins is through the fermentation of recombinant microorganisms engineered to express genes encoding the peptide or protein of interest. However, recombinant microbial peptide production has a number of obstacles to be overcome in order to be cost-effective. For example, peptides produced within recombinant microbial host cell are often degraded by endogenous proteases, which decrease the yield and increase the cost of production. Additionally, microbial production of smaller peptides in high yield may be adversely affected by size and the amino acid composition of the peptide. This is especially evident when the peptide of interest is soluble under typical physiological conditions found within the production host.
One way to mitigate the difficulties associated with recombinant peptide production is the use of chimeric genetic constructs encoding heterologous proteins. Also called fusion proteins, the heterologous proteins typically comprise at least peptide/protein of interest linked to at least one peptide tag. Linking the protein of interest [POI] to the peptide tag, also called solubility tag or inclusion body tag can make the POI insoluble. The peptide tag may be used to assist protein folding, post expression purification (e.g. His tags), protein passage through the cell membrane as well as to protect the peptide or protein from the action of degradative enzymes found within the cell.
Expressing a peptide in an insoluble form by fusing it to a solubility tag-even when the peptide is soluble at normal physiological conditions—facilitates recovery and protects the peptide from degradation. The fusion protein may include at least one cleavable peptide linker separating the solubility tag from the peptide of interest to facilitate subsequent recovery of the POI from the fusion protein. The fusion protein may include a plurality of inclusion body tags, cleavable peptide linkers, and regions comprising the peptide of interest.
Increasing the expression level of the gene encoding the POI can increase the POI yield, e.g., by chromosomal integration of multiple copies of the gene, use of a stronger promoter, and/or by using a high copy expression plasmid. However, the use of high copy plasmids often places an undesirable metabolic burden on the host cell.
Mutations to periplasmic proteases have been reported to increase recombinant antibody fragment accumulation in the E. coli periplasm (Chen et al., Biotech Bioengin (2004) 85 (5):463-474. Even though single gene knockout libraries are available for E. coli (Baba, T., et al., (2006) Mol. Syst. Biol. 2: article 2006.0008), down-regulating or disrupting specific genes or combinations of genes in E. coli that significantly increase heterologous peptide production are not as well known.
The problem to be solved is to provide Escherichia cells comprising knockout mutations to endogenous genes that increase the amount of a heterologous peptide produced within the host cell and methods of increasing recombinant production of a peptide of interest in the recombinant host cell comprising the down-regulated and/or disrupted endogenous gene(s).